Title

Author

Date of Award

5-1-2002

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

Abstract

The phosphorylation and desensitization of G-protein coupled receptors involve several protein kinases. Two of the major ones are protein kinase A (PKA) and protein kinase C (PKC). It has been reported that gravin, an A-kinase anchoring protein, plays a role in regulating p2-adrenergic receptor desensitization, resensitization, and internalization by recruiting both PKA and PKC, phosphatase, |3-arrestin, and clathrin to the receptor. Similarities between the regulation of p-opioid receptor and p2-adrenergic receptor suggest that a similar complex involving gravin may play a role in regulating p- opioid receptor signal transduction. To investigate this possibility, the current study examined the distribution of gravin, p-opioid receptor and p2-adrenergic receptor in SH- SY5Y cells as well as in AN3 CA cells cotrans fected with gravin and p-opioid receptor or p2-adrenergic receptor vector constructs. This study also examined the distribution of gravin, p-opioid receptor and p2-adrenergic receptor transgene in transfected AN3 CA cells after the agonist stimulation. Our data showed that gravin is distributed along the membrane of SH-SY5Y cells and partially colocalized with the p-opioid receptor and the p2-adrenergic receptor. The gravin transgene was also concentrated along the membrane of AN3 CA cells. Immunofluorescent microscopy showed extensive colocalization of gravin and either the p-opioid receptor transgene or the p2-adrenergic receptor transgene before agonist treatment. However, after agonist treatment, the p-opioid receptor and p2- adrenergic receptor transgenes translocated from plasma membrane to a perinuclear location, but there was no redistribution of grav in. The current study provided evidence that the recombinant gravin and the receptor proteins were expressed and distributed in a similar way as the native proteins. This validated their use in studying p-opioid receptor and p2-adrenergic receptor signal transduction and the role of gravin in regulating the function of these receptors. The current study also provided evidence that gravin might not interact with the receptors during the internalization of the receptors.